Homocysteine Intracerebroventricular Injection Induces Apoptosis in the Substantia Nigra Cells and Parkinson's Disease LikeBehavior in Rats

Parkinson's disease is a degenerative disorder of the central nervous system. The motor symptoms of Parkinson's disease result from the death of dopamine-generating cells in the substantia nigra, a region of the midbrain; the cause of this cell death is unknown. Homocysteine (Hcy) is a non-protein amino acid. It is a homologue of the amino acid cysteine. The elevated levels of homocysteine in plasma have been associated with a number of disease states. Hcy (2 µmol / µl) was injected intracerebroventricular (i.c.v) in rats, five days later, the locomotor activity was measured with open field apparatus, Also apoptosis was investigated in substantia nigra cells by immunohistochemical analysis. Hcy could decrease locomotor activities significantly in rats as well as it could induce apoptosis in substantia nigra cells. These results suggest that Hcy is a neurotoxic metabolite and may induce cell death in some nuclei in the brain.

arkinson's disease (PD) is the second most common neurodegenerative disorder after Alzheimer's disease (AD) (1) Also, it is progressive and leads patients to a debilitating condition and includes as well extensive dopaminergic neuron degeneration in the substantia nigra pars compacta (2) and the other subcortical nuclei with motor and non-motor symptoms. Motor symptoms are discriminated by hypokinesia, rigidity, tremor, and postural imbalance (3) and non-motor symptoms including autonomic dysfunction, neuropsychiatric problems, and sensory and sleep difficulties, which are common. Homocysteine is considered a risk factor for multiple neurological disorders including AD and PD (4,5,6). Homocysteine (Hcy); a sulfur containing amino acid derived from the metabolism of methionine, is an independent risk factor for cardiovascular disease (7 (10). In spite of many researches in this area, the molecular mechanism of homocysteine-induced neurotoxicity has not been completely established at present.

D-L-Homocysteine was purchased from
Sigma-Aldrich, Germany. Ketamine and xylazine were obtained from ALFASAN Co, Netherlands. Immunohistochemical and behavioral analyses were performed five days after Hcy injection in rats.

Intracerebroventricular (i.c.v.) Injection
For i.c.v. drug administration, the rats were anesthetized using ketamine ( which was attached to a 5-µl Hamilton syringe.
Behavioral and histo chemical analyses were performed five days after Hcy injection in rats.

Measurement of Locomotors Activities
The effects of Hcy on the rats' behavior were studied by an open-field apparatus. All experiments were carried out 5 days after Hcy (intracerebroventricular) injection in rats. The measurement was started 3 min after the placement of animals into the monitor in a quiet isolated place with a dim light. After the rats were injected (i.c.v.) with Hcy once a day, they were placed in the locomotors activity monitor (Ethovision-XT; Noldus, Netherlands). The changes in motor activity of the animals were measured. Total distance and velocity were determined. The locomotors activities were determined for 40 min in 6 th day, 5 days after Hcy i.c.v injection in rat. (11).

Brain Histopathological Analysis
At the end of the behavioral experiments, the rats were deeply anesthetized with a high dose of ketamine (150 mg/kg) and perfused through the ascending aorta with 50-100 ml of 0.9% saline

Bax/Bcl-2
To determine whether homocysteine leads to changes in Bcl-2 family protein levels in the rat brain, we examined the Bcl-2 and Bax protein immunostaining. As shown in fig. 2 fig. 4, the Bax/Bcl-2 ratio was calculated for SN tissue as explained above.
The results showed that in control group, this ratio is lower than homocysteine group which were significantly increased (F (1, 10) = 97.93, p<0.001).

Discussion
The aim of this study was to investigate the neurotoxic effect of Hcy in SN cells in the rat brain,   This result is consistent with that of Lee et al. who reported a decrease in the level of locomotor activity after acute homocysteine injection (11).
Literature data indicate that Hcy is toxic to neuronal cells (14). Moreover, hyperhomocysteinemia has been implicated in neuronal plasticity and neurodegenerative disorders in human study (5). The concentration of Hcy in the brain and cerebrospinal fluid is elevated in several neurological diseases in human and experimental animals (5,15). Numerous studies have reported that homocysteine is elevated in Levodopa therapy for PD patients and suggested a substantial role of homocysteine in causing various neurotoxic effects (16).
In the present study, we investigated the molecular response of the rat brain with immunohistochemical methods. The results showed that the expression of apoptosis regulatory proteins Bax and Bcl-2 would be altered by homocysteine which elevated the Bax/Bcl-2 ratio in favor of apoptosis.
Apoptosis is a morphologically and biochemically well characterized form of programmed cell death to remove the unnecessary or damaged cells in various situations (17). Apoptosis leads to cell death and differs from necrosis by distinct morphologic and biochemical features (18).
A key factor in determining cell death or survival following apoptotic signals is the relative expression of Bax and Bcl-2 proteins. The interactions between these pro-apoptotic and antiapoptotic proteins regulate the release of cytochrome c and the propagation of apoptotic cascade (19). The role of these apoptotic proteins in adjusting the number of neural precursors and post mitotic neurons during the development of nervous system has been established (20).
The results of the present study showed that Hcy was neurotoxic for rats. Histopathological results revealed that 5 days after Hcy (i.c.v.) injection, Bax level was significantly increased while Bcl-2 level was dramatically decreased in the substantia nigra in comparison to the vehicle and control groups (Figs. 2 and 3). It has been reported that hyperhomocysteinemia causes increase in proapoptotic Bax levels and decrease in anti-apoptotic Bcl-2 levels in the rat brain (21).
Our results suggested that Hcy might induce apoptosis and cell death in rat brain. Hcy may induce oxidative stress and produce ROS that attack all biological macromolecules (e.g. proteins, DNA and lipids). It is suggested that Hcy may be a risk factor for AD and PD.